Public Antibiotic resistance is common in bacteria associated with humans. This characteristic is often used to identify sources of environmental contamination by determining bacterial growth in the presence of antibiotics. A DNA element called an integron, which can capture, mobilise and express genes allows bacteria to be resistant to ... antibiotics. This mechanism can move antibiotic resistant genes to individuals within a species and between different species. The discovery of integrons has created novel approaches for tracking bacterial contamination. We aim to apply these tools to determine the distribution of antibiotic resistance genes from human sewage through the Antarctic environment.

Project objectives: 1) To track the dissemination of antimicrobial resistance gene cassettes from human sewage though the Antarctic environment by determining the distributions of integron associated antimicrobial resistance gene cassettes in sewage, sediment, ice and two marine invertebrate species.

3) To determine if lateral gene transfer is occurring between human sewage micro-organisms and an abundant Antarctic micro-organism (Pseudomonas).

4) To determine phenotypic and genotypic profiles of Escherichia coli in contaminated sewage areas to provide a system for tracking the distribution of these organisms to nearby Antarctic ecosystems.

Taken from the 2008-2009 Progress Report: Progress against objectives: 1) In working towards determining the dissemination of antimicrobial gene cassettes from human sewage analyses began using core samples collected pre-grant during 07/08 season. Screening of environmental DNA extracted from the 77 core samples (8 overall sites) identified antibiotic resistance mechanisms in 21 of 54 samples (38%) from the sewage impacted site (Shannon Bay) and 3 of 21 samples (14%) from the non-impacted site (O'Briens Bay). During 08/09 season samples of ice, sewage and sediment were obtained from Casey Station. These samples will be collected and transferred to Macquarie University in May, and screening of samples for antimicrobial gene cassettes will be the next step in determining the distribution of antibiotic resistance mechanisms in the Antarctic environment.

2) We have begun characterising gene cassettes from a sub-set of positive samples (3 from each site) from Shannon and O'Briens Bay (objective 1). For each sample DNA sequencing will be used to determine gene array and the types of antibiotic resistance genes that are present. This data will establish if the resistance mechanisms are from human sources, and provide the basis for screening subsequent samples (ice, sewage, sediment and marine invertebrates) for resistant cassettes to achieve objectives 1, 2 and 3.

3) This objective requires further data from objectives 1, 2 and 4 before progressing with the experimental approach.

4) Characterisation of E. coli will be performed on ice, sewage and grab samples collected during 08/09 season. This will include culture of these organisms and subsequent PCR. Reagents have been purchased and experimental approach determined.

Taken from the 2009-2010 Progress Report:

Progress against objectives:

1) Sediment, ice and marine invertebrates (Abatus and Laturnula) from Casey were screened for the presence of clinical integrons classes 1, 2 and 3. Very few integrons were identified in sediments, none in invertebrates but class 1 and class 2 integrons were identified in ice samples collected across sewage plume. Positives are currently being tested to determine gene cassette array. Additional samples of sediments, marine invertebrates and faecal samples from wildlife were collected in summer season of 2010 from Davis, these will be analysed over coming months for integrons classes 1,2,3 and additional characterisation performed on positives.

2) Integron diversity / detection in Casey samples was low. Cassette PCR has identified a range of cassettes in detected class 1 and class 2 integrons. Sequences from these sample are currently being analysed but preliminary results have identified a potentially novel cassette that may encode for antibiotic resistance.

3) Sediment samples collected at Davis Summer 09/10 are to be used for this project goal to be undertaken in project year 3.

4) An E. coli library of 463 isolates was established at Davis summer 09/10. The isolates were from sewage, water column, sediments, marine invertebrates and local wildlife species. The library will be genotyped over the coming months.

Taken from the 2010-2011 Progress Report: Progress against objectives: 1) Total environmental DNA was extracted from a total of 294 samples representing the 48 sediment cores that were collected for PCR analysis. Slicing of the sediment cores produced between four and seven sub-samples per site. PCR screening for integrase class 1 was performed on all samples and resistance determinants were detected at 9 sites. Positive samples were confirmed by gene cassette screening. The amplicons of the gene cassette screening contained multiple bands indicating several constructs may be present in sediment. DNA was extracted from 445 cryopreserved bacterial isolates followed by subsequent PCR screening. Of the 445 isolates 366 were PCR competent as determined by positive 16S rDNA screening. These isolates were obtained from a range of sources (sewage, sediment, and the marine invertebrates Abatus and Laturnula). The 366 samples competent samples were screened for antibiotic resistance mechanisms with 76 samples positive. These samples were positive for integrase class 1 and gene cassette PCR. Positive samples were derived from various sources including the water column, STP sediment and Laternula depauparate. The amplicons from gene cassette screening ranged from 450 bp to approximately 2000 bp. The gene cassette amplicons were purified for DNA sequencing to achieve objective 2. We have also performed T-RFLP on the 294 sediment samples to examine diversity between impacted and non-impacted sites. We are currently performing community analysis on these samples.

2) DNA sequencing was performed on 46 isolates. We have identified genes that encode for the resistance of three antibiotics, streptomycin and spectinomycin (genes AADa1 and AADa5) and trimethoprim (gene dhfra17). Of the 46 isolates sequenced AADa1 was present in 35 isolates, dhfra17 and AADa5 in 2 isolates and the remaining. The constructs are variable indicating multiple human sources. The constructs are also commonly found in human clinical settings.

3) This objective has been compromised due to freezing on samples on return trip from Davis. Viable microbial cells are required.

4) We performed a phylogenetic analysis on the E. coli library with 353 isolates positive for the test. The isolates can be grouped into 4 strains A (117), B1 (40) B2 (157) and D (39). We have performed a fingerprint analysis on all strains to determine further diversity and analysis is ongoing. We will compare these results with the isolates that were positive for resistance determinants to identify any patterns in strain survival and dispersion.

Quality
The values provided in temporal and spatial coverage are approximate only.

Taken from the 2008-2009 Progress Report: Field work: 1) Samples were collected at Casey during the 08/09 season by J. Stark (Complete) a) effluent (2 x 50-100 ml) from waste water treatment plant ... b) Ice samples (50-100g) in a transect across the sewage plume of waste water outlet c) Water samples (2 s 50 ml) from five points close to sewage outfall in Shannon Bay and five from O'Briens bay d) Sediment grab samples from Shannon Bay and five from O'Briens bay

2) Samples collected from Casey in 08/09 season are in storage in Kingston and will be collected by Michelle Power and transported to Macquarie University for analyses (May 2009)

Laboratory activity/analysis: 1) Preliminary screening using common PCR method of core samples from 07/08 season for Antibiotic resistance genes (completed). 2) Positives from core samples undergoing characterisation by sequencing of Gene cassettes with six samples completed by October 2009. 3) Three further tests for antibiotic resistance genes will be performed on core sample DNA (completion July 2009) 4) Screening by T-RFLP to determine microbial diversity at variable depths has begun on core samples. This component will be completed and submitted for publication by September 2009. 5) Screening for antibiotic resistance cassettes and analysis of gene cassettes from sediment, ice water and sewage collected from 08/09 season will begin in July 09 and completed by June 2010 (Honours Project) 6) Culture of E.coli from sediment, sewage and ice samples will begin on collection of samples from Hobart (May 2009) 7) Screening of marine invertebrates will begin in late 2009 using data from other samples to design optimal experimental approach.

Taken from the 2009-2010 Progress Report:

Variations to work plan or objectives:

No changes have been made, although we have a significantly greater number of samples obtained from the summer 09/10 than anticipated in original grant application. This may require prioritising of goals based on achieving goals 1, 3 and 4 as outlined above. The minimal identification of integrons in Casey samples may lead us to readdress goal 3 but this will be dependent on outcomes of analysis of E. coli and sediments from Davis ie if low diversity / occurrence of integrons in enviroment can we modify goal 3 in line with these outcomes.

Field work:

Fieldwork undertaken at Davis V3-V4 2010. An Escherichia coli library containing 463 E. coli isolates preserved in glycerol and stored at minus 80oC was established. The E. coli were obtained from samples described above, and from water column samples provided by Jim Smith. The library will be used to determine phenotypes and genotypes of E. coli in the Antarctic environment and relationship to those in sewage. This component represents milestones for end of project year two although we have obtained an excess of some 363 cultures that were not anticipated in original grant. Additional funding will need to be sort to complete analysis of additional samples. For the 100 samples expected genotyping and virulence factor analysis will be completed by September 2010.

Sample library: Sediment cores from 24 dive sites and beach area at Davis, faecal material from seals and penguins and intestinal contents from Abatus have been allocated barcodes and returned to Australia for subsequent genetic testing. These samples require DNA extraction, diversity determination and screening for class I integrons that will be completed by October 2010. Characterisation of resulting gene cassettes will be performed in following months.

All samples are currently in Hobart and details of customs clearance or locations have not been provided to Investigator to date.

Laboratory activity/analysis:

LABORATORY ANALYSIS: MACQUARIE UNIVERSITY

Sediment cores (77) grab sediments (4) and ice (10) were screened for integrons. Of this 15 samples went onto further analysis for gene cassette arrays, cloning and sequencing. The sediment cores were also tested for diversity using 16S rDNA ribotyping. The general trend was that diversity decreased with and community structure altered with depth. Communities in ice samples covering sewage plume and top layer sediments were similar suggesting that organisms were making their way into bay and settling into sediments. The data collected and sequencing is still undergoing analysis with final results for testing of Casey sample anticipated by July 2010.

LABORATORY ANALYSIS: DAVIS 2010

Outfall testing: four samples from the outfall were tested for coliforms using Colilert quantitray system. Bacteria counts for each sample were greater than 2.6 x 106 E. coli / 100ml of sewage outfall water. Sediments testing: Replicate core samples from 2 plots within 24 sites were provided by dive team. Samples were tested for E. coli using Colilert quantitray system. Sediments from seven sites tested positive for coliforms / E. coli. Counts ranged between 1 and 8 E. coli cells / ~ 10g sediment. The sites that tested positive were STP5, STP6, STP10a, STP10b, STP10c, STP10d and STP13a. Marine invertebrates testing: Intestinal contents from 30 Abatus were screened for E. coli using Colilert quantitray system. Samples were obtained from STP4, STP5, STP10, STP13a, Old wallow, outfall and STP2. These samples were provided by the dive team and Jake Van Ooestrom. The results for coliform testing of Abatus were hampered by false positives, which has been attributed to two factors 1) chitin present in copepods may be causing conversion of enzyme in media or 2) a bacteria endemic to Antarctic waters may be utilizing enzyme. Our aim is to investigate this further but preliminary findings support the chitan suggestion. Copepods were incubated with colilert media for 24 hours, this resulted in conversion of media to the blue colour used to indicate E. coli. Faecal sample testing: Faecal samples were obtained from a range of vertebrate wildlife species to enable comparison of E. coli phenotypes from sewage outfall, sediments and the water column. Samples were obtained from elephant seals, weddell seals and Adelie penguins at a range of sites. In total 57 faecal samples were tested from the above species. Isolates have been added to library and samples returned to Australia for further testing (see below).